In phacoemulsification, ultrasound induces hydroxyl radical (center dot OH) formation, damaging corneal endothelium. Whether H-2 can prevent such oxidative damage in phacoemulsification was examined by in vitro and in vivo studies. H-2 was dissolved in a commercial irrigating solution. The effects of H-2 against center dot OH generation were first confirmed in vitro by electron-spin resonance (ESR) and hydroxyphenyl fluorescein (HPF). ESR showed a significantly decreased signal magnitude, and fluorescence intensity by oxidized HPF was significantly less in the H-2-dissolved solution. The effects of H-2 in phacoemulsification were evaluated in rabbits, comparing H-2-dissolved and control solutions. Five hours after the procedure, the whole cornea was excised and subjected to image analysis for corneal edema, real-time semiquantitative PCR (qPCR) for heme oxygenase (HO)-1, catalase (CAT), superoxide dismutase 1 (SOD1), and SOD2 mRNA, and immunohistochemistry. Corneal edema was significantly less and the increases in anti-oxidative HO-1, CAT and SOD2 mRNA expressions were significantly suppressed in the H-2 group. In addition, corneal endothelial cell expressions of two oxidative stress markers, 4-HNE and 8-OHdG, were significantly lower in the H-2 group. In conclusion, H-2 dissolved in the ocular irrigating solution protected corneal endothelial cells from phacoemulsification-induced oxidative stress and damage.